1. Field of the Invention
The present invention is directed to the separation of nucleic acids using nonporous polymeric beads. More specifically, the invention is directed to the chromatographic separation of both single strand and double strand nucleic acids by chromatography using chromatographic columns containing alkylated nonporous polymer beads.
2. Discussion of the Background
Nucleic acid separations have become a focus of scientific interest and numerous groups of researchers have been attempting to achieve improvements of various technical aspects in this area. Anion exchange separations together with ion pairing/reverse phase chromatography are among the most frequently used methodologies for the separation of nucleic acid species.
In European patent application (EP 0 507 591 A2), W. Bloch showed that, to a certain extent, a length relevant separation of DNA fragments was possible on nonporous anion exchangers with tetramethylammonium chloride (TMAC) containing eluents. However, as shown in FIG. 5 of this patent application, fragments with 458 and 504 base pairs (difference of 46 base pairs) elute at the same time and are not separated. Although, on the other hand it was possible to separate fragments differing only by 34 base pairs (fragments with 434 and 458 base pairs).
Additionally, an addition of TMAC causes a significant, general decrease in resolution. Also, because of the ion strength gradients necessary for achieving separations by Bloch's method and the resulting high nonvolatile salt concentrations, any subsequent examination and measurements on the separated fragments are not possible.
The method for separating DNA fragments on anion exchange materials, carrying trimethylammonium groups, reported by Y. Ohimya et al (Y. Ohimya et al, Anal. Biochem., (1990), 189:126-130) has the same drawbacks as the method utilizing anion exchangers with diethylaminoethyl groups for the separation of DNA fragments (Y. Kato et al, J. Chromatogr., (1989), 478:264). A separation strictly dependent on the size of DNA fragments is impossible in both cases. Furthermore, any subsequent measurements with the separated fractions are impossible due to high salt concentration of these fractions.
An important disadvantage of anion exchange separations of double stranded nucleic acids is the differing retention behavior of GC- and AT- base pairs. This effect makes a separation according to molecular size impossible. General utility of anion exchange for nucleic acid analyses is thus strongly reduced. Another important drawback of the anion exchange methodology is the necessity to use ionic strength gradients to achieve elution over the entire range of nucleic acid molecular weights. Strong contamination of eluting peak zones by salt, due to the ionic strength gradients, makes subsequent investigations of DNA molecule fractions very difficult.
There are also serious drawbacks with applications of reverse phase/ion pairing chromatography to separations of double stranded DNA fragments, at least with the current version of the procedure introduced by Eriksson et al. Most importantly, the relatively low separation efficiency of this methodology leads to insufficient resolution and to low recoveries for very short restriction fragments. Typical analytical run times with ion pairing/reverse phase chromatography are in the range of one to several hours (see S. Eriksson, G. Glad, P. A. Pernemalm, E. Westman J. Chromatogr, (1986), 359:265-274).
Some limited separations using nonporous polymeric packing beads were described by J. Thompson in Biochromatography, (1986), 1:16; (1986), 1:22; (1986), 1:68; (1987), 2:4).
Huber et al were the first to fully discuss the utilization of non-porous polystyrene beads as a stationary phase for reverse phase/ion pair chromatography. The disadvantages of the method used by Eriksson et al were thought to center around the use of porous silica beads for the reverse state stationary phase (C. G. Huber, P. J. Oefner and G. K. Bonn, J. Chromatogr., (1992), 599:113-118). Huber et al were able to show that separations of single stranded nucleic acids could indeed be improved by changing over from the silica based reverse phase material to nonporous polystyrene beads. Additional improvement was accomplished by inclusion of a polyvinyl alcohol into the polystyrene nonporous beads. Inclusion of polyvinyl alcohol was accomplished by addition during one of the synthetic steps of the procedure leading to the nonporous polymeric beads.
On the other hand, Huber et al were unable to achieve the same kind of improvement for the double stranded nucleic acids with their polymeric beads. More specifically, with the double stranded molecules, they observed an insufficient resolution for all analytes having more than 150 base pairs.
A need continues to exist for chromatographic methods for separating nucleic acids with improved separation efficiency and resolution.